Ewing sarcoma is the second most common bone and soft tissue malignancy in children and adolescents. The five-year survival of Ewing sarcoma patients is 70%, and falls to less than 20% for those with metastatic disease, a situation that has not improved despite many years of increasingly intensive chemotherapy regimens. Survivors of Ewing sarcoma often suffer long-term deleterious effects of treatment. An immediate need exists for the development of effective, targeted therapies with decreased toxicity. Molecularly, Ewing sarcoma is characterized by one of several reciprocal chromosomal translocations that fuse a FET family protein with an ETS transcription factor, the most common of which is EWSR1-FLI1. The EWSR1-FLI1 fusion protein is completely specific to Ewing sarcoma tumor cells, and thus presents an ideal target for novel therapeutics. To date, however, no such targeted therapy has become clinically available, owing to 4 major knowledge gaps. First, there is a dearth of robust in vitro model systems for functional studies of EWSR1-FLI1. Knockdown studies in Ewing sarcoma cell lines can give inconsistent results, and few primary cell types tolerate exogenous EWSR1-FLI1 expression. Second, the protein encoded by the EWSR1-FLI1 fusion is a transcription factor, and there is widespread perception that such proteins are ?undruggable?. Third, lead candidates of small-molecule screens reported to date have either not been shown to have a mechanistic link to EWSR1-FLI1, or fail to replicate the phenotype of Ewing sarcoma cells depleted of EWSR1-FLI1 activity. Fourth, the field has lacked relevant animal models for functional genomics and preclinical testing. Developing targeted therapy for Ewing sarcoma will require an integrated approach executed by an experienced team. Our strategy is based on several fundamentals, namely 1) systems to specifically deplete and reconstitute the EWSR1-FLI1 protein in cells; 2) an understanding of the structural biochemistry of low-complexity (LC) domains found in EWSR1 and related proteins; and 3) novel in vivo models of Ewing sarcoma for functional genomics and drug testing. Standing on this foundation, we will perform biochemical and genetic experiments to identify the binding partners and functional effectors of EWSR1-FLI1 and dissect its mechanisms of transcriptional activation. We will use our experimental systems to conduct phenotypic and target-based high- throughput screens. Finally, we are well-positioned to perform the work required to transform a compound with promising initial activity into a preclinical drug lead. Our approach to drug discovery is built upon medicinal chemistry coupled with secondary assays that maximize potency and specificity of lead compounds. We will perform target identification for lead compounds using strategies our team has successfully used for several agents currently in active preclinical development. A dedicated preclinical pharmacology core, highly integrated with the projects and with the high throughput screening core and medicinal chemistry core, will ensure optimization of delivery, PK/PD and toxicity properties of the most promising lead compounds.